*4.13. Antibacterial Activity of CS/PVOH/HNT and CS/PVOH/TO@HNT Films*

Antibacterial properties of the films were investigated by utilizing the agar diffusion method against Gram-negative bacteria *Escherichia coli* (ATCC 25922), *Salmonella enterica* subsp. enterica (DSMZ 17420), Gram-positive bacteria *Staphylococcus aureus* (DSMZ 12463), and *Listeria monocytogenes* (DSMZ 27575). The tested foodborne microorganisms were obtained from the Institute of Technology of Agricultural Products, ELGO-DEMETER, Lykovryssi, Greece.

Fresh cultures of the bacterial strains were prepared in Mueller Hinton Broth. The cultures were inoculated at 37 ◦C for 24 h in order to achieve a range of 107–10<sup>8</sup> CFU mL−<sup>1</sup> . After that, the bacteria were swabbed on Mueller-Hinton agar dishes by rotating the plate every 60◦ to ensure consistent growth.

The tested films were cut into 6 mm diameter discs by a circular knife and were placed on a Mueller-Hinton inoculated plate. The dishes were incubated at 37 ◦C overnight. The diameter of the inhibitory zones, and the contact area of the discs with agar surface, were measured. The experiment was performed thrice.
